Bay-region diol epoxide (DE) metabolites of carcinogenic polycyclic aromatic hydrocarbons are believed to initiate cell transformation by covalent modification of DNA. Two diastereomeric DEs, each of which exists as a pair of enantiomers, are formed metabolically in mammals: DE-1, in which the benzylic hydroxyl group and epoxide oxygen are cis, and DE-2, in which these substituents are trans. The primary targets in DNA for these DEs are the exocyclic N-2 and N-6 amino groups of deoxyguanosine (dG) and deoxyadenosine (dA), respectively. The goal of our research is to elucidate the relationships between the physical structures of DNA adducts formed from specific DEs and their biochemical processing with purified enzymes and in intact cells. A highlight of the laboratorys synthetic work this year has been the development of improved methods for the preparation of both cis and trans opened DE adducts at N-2 of dG as their phosphoramidites, using a novel blocking/deblocking protocol for O-6 of dG. Previously, these trans opened dG adduct phosphoramidites had required a more laborious, multi-step route, and their cis opened counterparts were essentially inaccessible synthetically. Availability of methods for the construction of DE-adducted oligonucleotides of defined adduct stereochemistry and nucleotide sequence makes possible the systematic study of effects of their structural features on mutations that result from DNA replication past the adducts. Last year, we reported the results of site-specific mutagenesis experiments in M13mp7L2 bacteriophage using eight 16-mer oligonucleotides containing dA adducts corresponding to trans opening of each enantiomer of the bay-region DE-1 and DE-2 from benzo[a]pyrene (BaP). These adducts were incorporated at the bracketed positions in two different sequence contexts derived from the supF gene: Context I, 5-TTT[A]GAGTCTGCTCCC-3, and Context II, 5-CAG[A]TTTAGAGTCTGC-3. We have now extended these mutagenesis studies to the cis opened BaP DE dA adducts, as well as the analogous cis and trans opened fjord-region benzo[c]phenanthrene (BcPh) DE dA adducts in the same two sequences. In a given sequence context, differences in overall mutagenicity between individual BaP adduct isomers were relatively small, and there was no clear relationship between stereochemistry and mutagenicity for adducts derived from this hydrocarbon. In contrast, the configuration at the site of attachment of each BcPh DE isomer to N-6 of dA had marked effects on mutagenicity, and constituted the dominant stereochemical feature influencing the mutagenic activity of these adducts. All the BcPh adducts with R configuration at the point of attachment of the hydrocarbon to N-6 were very poor mutagens in Context I (mutation frequencies less than 1%), whereas the adducts with S configuration at this carbon atom exhibited mutation frequencies from 10-32% in this sequence. Although this stereochemical effect was less pronounced in Context II, it is notable that an adduct with the S configuration was the most mutagenic of all the BcPh DE dA adducts in both sequences (mutation frequency 32% in Context I and 57% in Context II). Topoisomerases are DNA processing enzymes that relax supercoiling and relieve torsional strain by forming reversible DNA cleavage complexes in which a tyrosine in the enzyme becomes covalently bonded to a 3-phosphate group, and a new 5-hydroxyl group is generated at the cleavage site. Certain anticancer drugs such as the camptothecins inactivate topoisomerase I (top1) by trapping the cleavage complex and preventing religation of the cleaved DNA. Many of these drugs are thought to be minor groove binders. We have investigated the effects of cis and trans opened N-2 dG adducts of the (+)- and (-)-enantiomers of BaP DE-2 in a short duplex oligonucleotide (22-mer) that is a substrate for top1. These hydrocarbon adducts provide unique probes for investigating the mechanism of top1 inhibition, since, unlike the camptothecins, they are covalently bound to the DNA, and their conformations in DNA are predictable from prior NMR studies: the trans opened dG adducts have the aromatic moiety oriented in the minor groove, whereas the cis opened dG adducts have the aromatic portion intercalated into the helix. These BaP DE adducts, when located on the dG that would have a 5-hydroxyl group after breaking the phosphodiester bond normally cleaved by top1, inhibit cleavage of this bond and induce DNA cleavage at other phosphodiester bonds in the sequence. The trans (minor groove) adducts were more effective than the cis (intercalated) adducts as inducers of a new cleavage site at the third phosphodiester bond 5 to the adduct. Of the four adducts studied, the trans opened dG adduct derived from (+)-BaP DE-2 was the most active in inducing cleavage by top1. To our knowledge, this constitutes the first example at the molecular level of a clear enhancement of potential DNA damage by this major adduct of the most carcinogenic BaP DE isomer as compared with the other three, stereoisomeric BaP DE-2 dG adducts.